The expression of globin genes is controlled at several levels in vivo. Negative as well as positive regulation of globin genes is crucial for their correct developmental regulation. It has been shown that two silencer regions upstream of the b-globin gene and a common protein that binds to both of them, termed beta protein 1 (BP1), participate in the negative regulation of transcription. BP1 binds within silencer I at ?530 bp and within silencer II at ?300 bp relative to the cap site (+1) of b-globin promoter. High mobility group protein HMG-I (Y) binds to and bends the DNA at or near the BP1 binding site in Silencer I but not in Silencer II. HMG-I (Y) may facilitate the binding of BP1 and possibly other repressor proteins in this region. Similarly, HMG1, another high mobility group protein, binds to and bends the DNA near the BP1 binding site in silencer II. To further investigate the mechanism of the negative regulation of b-globin gene expression by BP1 we constructed a number of clones of b-globin gene sequence (690 bp region) containing b-globin promoter and upstream promoter element that harbors two BP1 binding sites. As silencer II does not overlap HMG binding sites, we generated multiple mutations at this BP1 binding site using site-directed mutagenesis technique. 690 bp region of b-globin gene promoter was linked to an enhanced green fluorescent protein (E-GFP) reporter. K562 erythroleukemia cells, which express BP1 and lack detectable levels of b globin mRNA, were efficiently transfected with E-GFP constructs. In the 690 bp region of b-globin promoter, these constructs contain one of the following: wild type sequence of silencer II; its mutated version; or a mutated control sequence (located slightly upstream of silencer II). We analyzed E-GFP expression in K562 cells using flow cytometry and digital fluorescent microscopy. Quantitative fluorescence intensity analyses of digital images and flow cytometry data showed at least 10-fold increase in b-globin promoter activity of mutated silencer II construct as compared to wild type or mutated control constructs. BP1 is a member of the homeobox gene family and belongs to the subfamily called Distal-less (DLX), genes important in early development. BP1 shares almost complete sequence identity with DLX7 from nucleotides 565 to 1251 (BP1 mRNA sequence is 1251 nucleotides). BP1 and DLX7 are isoforms of DLX4. There is very little information about DLX7 function and no information about the role of DLX4. Inhibition of DLX7 causes decreased expression of GATA-1 and c-myc genes in K562 cells expressing high levels of DLX7. We started experiments using RNA interference methodology and monitored siRNA-induced silencing of BP1 expression in K562 cells. To achieve maximal BP1 gene-silencing effect we will use double-stranded siRNA having a 21-nucleotide complementary sequence with BP1 coding region. We plan to simultaneously analyze levels of b-globin and BP1 gene expression using real time PCR. We have also initiated experiments to establish a transgenic mouse model for specific repression of adult beta-globin expression by the BP1 protein. The murine Dlx7 is homologue of human BP1. To alter developmental specificity of the human beta-globin gene, we introduced a mutated BP1 binding site (Silencer II) into the distal promoter of beta-globin gene sequence of 35 kb cosmid construct containing the micro-LCR (locus control region) and other essential elements of human beta-globin gene cluster. This construct and appropriate controls have been microinjected into the single cell mouse embryos. The use of the transgenic approach should permit the in vivo analysis of the role of BP1 silencing motif during the embryonic-fetal developmental stages.